This invention relates to nucleic acid vaccines and more specifically to Dengue nucleic acid vaccines.
Dengue (Den) viruses belong to the flavivirus genus of the family Flaviviridae and are of four serotypes, Den 1-4. Dengue viruses are positive strand RNA viruses which code for ten genes. The genes are translated as a polyprotein which is cleaved by host and viral proteinases. The virus envelope (E) protein is the major antigen against which neutralizing antibodies are directed. These antibodies have been shown to be capable of protecting against dengue virus infection1. The membrane protein also appears on the virion surface and is required for the proper processing of E.
Dengue viruses are transmitted primarily by the mosquito, Aedes aegypti, and are a major cause of morbidity and mortality throughout tropical and subtropical regions worldwide2. It is estimated that there are over 100 million cases, annually, of dengue fever3. Human dengue illnesses range from an acute undifferentiated fever to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). A primary infection usually causes dengue fever. The illness is generally mild and the person apparently acquires a life long immunity against the serotype of dengue virus causing the infection. However, if a person acquires a second dengue infection with a different serotype, the illness may be more severe and lead to hemorrhagic fever or shock syndrome, with a mortality rate between one and five percent. The increased severity of the secondary infection is caused by an immune enhancement phenomenon4. Immune enhancement begins when non-neutralizing antibodies, generated from the earlier infection with one dengue serotype, bind to but do not neutralize the virus causing the secondary infection. The Fc portion of the antibody in the virus-antibody complexes then binds to the Fc receptors present on mononuclear phagocytes of the immune system, resulting in enhanced infection of these cells. The enhanced infection leads to the release of cytokines that ultimately cause capillary leak and coagulopathy, the principal pathogenic mechanisms in DHF/DSS.
At present there is no approved vaccine for dengue viruses. The most effective dengue vaccine would elicit sustained protective levels of neutralizing antibodies against all four serotypes so as to avoid the possibility of immune enhancement in a vaccinated individual who might become secondarily infected with a different epidemic or endemic dengue of a different serotype.
Work in mice and primates with inactivated whole virus and at recombinant protein dengue vaccines has generally been disappointing because of the lack of sustained protective neutralizing antibodies induced. Vaccination with live attenuated dengue virus vaccines is another promising approach, but difficulties still remain in developing a product that is immunogenic and does not cause dengue fever-like side effects. DNA vaccines for dengue will offer substantial advantages over these other approaches in that sustained immunity can be achieved without the risk of dengue fever-like side effects or immune-enhancement.
A description of DNA inoculation was presented by Vical, Inc. (San Diego, Calif.)5. Vical demonstrated that one could inject a gene, (in the proper context of an eukaryotic expression vector), into the muscle of an animal and that the injected gene would be expressed. The expression vector must contain the proper eukaryotic transcriptional regulatory elements (promoter/enhancer and polyadenylation site) and a multiple cloning site so that once inside the cell of the subject the gene will be expressed. Once the gene is transcribed it is translated and processed to its mature protein product.
The basic DNA injection system offers great potential for vaccine development. One clones the genes which contain the desired antigenic determinates into an expression construct and injects the DNA into an animal. Since the proteins are translated and processed within the host cells, proper conformation of B cell epitopes on secreted proteins and induction of class 1 MHC-dependent immune responses should occur appropriately.
The technique of DNA injection is being used to develop vaccines against many pathogens including: Influenza6, HIV7, Hepatitis C8, Malaria9 and Herpes10.
It is an object of this invention to protect a subject or community against infection by dengue virus.
It is another object of this invention to provide protection against infection by dengue virus by using a nucleic acid vaccine.
It is an object of this invention to provide protection against more than one dengue virus serotype.
These and additional objects of the invention are accomplished by taking the envelope (E) and optionally, the membrane (PreM) genes of dengue virus, serotypes 1, 2, 3, and 4 and cloning them into eukaryotic plasmid expression vectors. The resultant plasmid DNAs (dengue DNA vaccines) are injected into a mammalian subject where in vivo the E proteins are expressed and subsequently recognized and processed by immune cells. This results in the generation of humoral and cellular immunity that is protective against dengue virus infections of each of the four serotypes.
We have used the DNA injection technology to develop DNA vaccines against the Den viruses. We have cloned genes from each of the four Den serotype viruses into expression vectors and have assessed their potential as Den virus vaccines. The Den genes which we have incorporated into our DNA vaccines include: 80-100% of the E gene; either alone or expressed with the PreM gene. The choice of these genes systems from publications which demonstrate that: E contains the virus"" major antigenic determinants11; not all of E is required to obtain a protective immune response12; and PreM affects the conformation of the produced E protein13. As Den virus DNA vaccines, some combinations of the above-listed genes yield better immune responses than others.
These and other objects, features and advantages of the present invention are described in or are apparent from the following detailed description of preferred embodiments.